A new route to trihydroxamate-containing artificial siderophores and synthesis of a new fluorescent probe

Article abstract of DOI:10.1016/j.bmc.2004.11.053

A fluorescent labelled artificial siderophore 1 was synthesized by coupling a 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative to the terminal amino group of a new trihydroxamate-containing amine 2, a ferrichrome-type siderophore that was obtained from tris

Full text of DOI:10.1016/j.bmc.2004.11.053

Bioorganic & Medicinal Chemistry 13 (2005) 1799–1803
A new route to trihydroxamate-containing artificial
siderophores and synthesis of a new fluorescent probe
H. Ouchetto,a,b M. Dias,a,* R. Mornet,a E. Lesuissec and J.-M. Camadroc
a
c
´ ´ ´
Chimie, Ingenierie Moleculaire et Materiaux d’Angers, UMR 6200 du CNRS, 2 Boulevard Lavoisier, 49045 Angers Cedex, France
Laboratoire de Chimie des Substances Naturelles et des Heterocycles, Faculte des Sciences-Semlalia,
b
´ ´
Universite Cadi Ayyad, Marrakech, Morocco
´
´
´ ´ ˆ ´
Laboratoire d’Ingenierie des Proteines et Controle Metabolique, UMR 7592 du CNRS, 2 Place Jussieu, 75251 Paris Cedex 05, France
Received 9 July 2004; accepted 25 November 2004
Available online 20 December 2004
Abstract—A fluorescent labelled artificial siderophore 1 was synthesized by coupling a 7-nitrobenz-2-oxa-1,3-diazole (NBD) deriva-
tive to the terminal amino group of a new trihydroxamate-containing amine 2, a ferrichrome-type siderophore that was obtained
from tris(hydroxymethyl)aminomethane. Compound 1 was shown to be a suitable tool for experiments on siderophore transport
and uptake processes in various organisms cells and particularly in Candida albicans cells.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
the influence of the various zones of the molecules on
the iron(III)-binding, recognition by the cells of Pseudo-
monas putida and penetration into these cells. It has been
shown that the R0 zone, can be modified without affect-
ing the iron binding, or the interaction with membrane
proteins for which the influence of the R substituents
is very sensitive5 (Scheme 1).
Although iron is one of the most abundant elements in
the earthÕs crust, in aerobic environments, it is found
as Fe(III) ion forming very insoluble oxides and hydrox-
ides. In order to overcome this low solubility and bio-
availability of iron, microorganisms synthesize strong
iron(III) chelators, called siderophores, and release them
into the environment where they selectively trap iron
and mediate its transport and deposition into the cell.1,2
In this paper, we report the synthesis of a fluorescent-
ferrichrome analogue 1 from a new trihydroxamate-con-
taining amine 2 (Scheme 1). The iron-binding part of
these molecules incorporates the glycyl chain (R = H),
which allows them to fully mimic the natural ferri-
chrome.4
Recent results have shown that iron uptake from
hydroxamate siderophores, and particularly ferri-
chrome-type siderophores, occurs in Candida albicans
and is mediated by one or more high-affinity transport
systems.3 For a better understanding of the mechanisms
of iron transport in C. albicans, we undertook the syn-
thesis of ferrichrome analogues. It is important from a
medical point of view to understand how C. albicans ac-
quires iron. Indeed, proteins that mediate iron transport
in C. albicans represent potential targets for the develop-
ment of anti-Candida therapies.
2. Results and discussion
2.1. Chemistry
Shanzer co-workers used R0-substituted pentaerythritols
as starting materials for the synthesis of ferrichrome
analogues. This strategy compelled them to reproduce
in each case the harsh building of the trihydroxamate
parts of the molecules (Scheme 1).4,5 We synthesized
amine 2 as a source of various amino-substituted deriva-
tives expected to have the ferrichrome siderophore
properties. This organic specificity is similar to those
Many ferrichrome analogues were synthesized by
Shanzer and co-workers4,5 with the aim of studying
*
Corresponding author. Fax: +33 24173 5405; e-mail: marylene.
dias@univ-angers.fr
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.053

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H. Ouchetto et al. / Bioorg. Med. Chem. 13 (2005) 1799–1803
OH
OH
OH
OH
OH
OH
N
N
N
N
OH
OH
OH
N
O
N
O
N
O
O
N
N
O
O
O
O
O
R
O
R
NH
NH
NH
R
NH
NH
NH
HN
O
O
HN
HN
O
O
O
O
O
O
NO2
O
O
O
O
O
O
O
O
O
N
O
N
R'
N
NH2
HN
O
N
O
Shanzer's analogues
O
1
2
Scheme 1.
of desferrioxamine B, a linear trihydroxamate-type side-
rophore with an amino-terminal group, from which
various derivatives have been synthesized.6–8 For the
preparation of 2, we opted for the synthesis of interme-
diate 3, a (poly-ether)amine, colloquially known as LinÕs
amine9 (Scheme 2). It was obtained in two steps from
tris(hydroxymethyl)aminomethane (ÔTrisÕ), a convenient
and inexpensive starting material, previously used for
the synthesis of dendritic macromolecules. Amine 3
was protected with the benzyl chloroformate (Cbz)
group, yielding 4. Subsequently, the removal of the ethyl
esters without conflict with the protecting group was ob-
tained, by using 1 M aqueous sodium hydroxide in eth-
anol at low temperature. Another way to obtain triacid
5 from ÔTrisÕ has been described by Cardona and Gaw-
ley,10 a three-step synthesis using an amino-polyether
tri-tert-butyl ester as an intermediate. The triacid 5 ob-
tained was treated with 3 equiv of hydroxamate-bearing
glycyl residue11 after activation of the carboxylic groups
by pentachlorophenolate groups yielding 7. The benzyl
chloroformate group was easily removed from 7 by
hydrogenation with Pd/C in ethanol and amine 2 was
obtained quantitatively.
O2N
N
N H
i
O2N
N
N
OH
O O
N
N
N
N
9
10
O
O
O
iii
ii
O2N
N
N
O N
O
1
O O
N
N
8
O
Scheme 3. Synthesis of NBD-ferrichrome analogue conjugate 1.
Reagents and conditions: (i) succinic anhydride, toluene/DMF (3:1,
v/v), D ; (ii) NHS, DCC, THF/DMF (4:3, v/v), 25 ꢁC; (iii) 2, THF/
DMF (3:6, v/v), 25 ꢁC.
lowed by esterification of the obtained acid 10 with
NHS.
2.2. Chelation of iron(III) by 1
Adding the NBD label to the amine 2 imparts fluores-
cent properties to the free iron carrier. Excitation of 1
in methanol (80%)/aqueous sodium acetate (20%) at
474 nm and monitoring of the emission spectra over
the 480–650 nm range reveal a single peak at 538 nm,
that is quenched upon loading of the carrier with ferric
iron (Fig. 1). In agreement with Shanzer and co-work-
ers,5 the observed fluorescence quenching of this ferri-
chrome analogue follows PerrinÕs model of static
quenching (Fig. 1 inset) and, as expected, the fluore-
The fluorescent-ferrichrome analogue 1 was prepared
by coupling amine 2 with the hydroxysuccinimidyl
ester-fluorescent probe 8 (Scheme 3). Compound 8 was
obtained in two steps from monosubstituted piperazinyl-
NBD5 9 by condensation with succinic anhydride fol-
O
i
ii
H2N
H2N
CN
H2N
OH
O
O
3
O
3
3
3
O
O
iii
iv
CBz HN
CBz HN
CBz HN
O
4
O
O
5
OH
3
3
OH
N
O
O
vi
v
CBz HN
OH
O
NH
CH3
O
OC6Cl5
3
3
O
6
7
O
vii
H2N
N
O
NH
CH
3 3
O
2
Scheme 2. Synthesis of trihydroxamate-containing amine 2. Reagents and conditions: (i) CH2@CHCN, aqueous KOH, dioxane; (ii) H2SO4, ethanol,
reflux; (iii) benzylchloroformate, K2CO3, dioxane; (iv) 1 N aqueous NaOH, ethanol/water (7:3, v/v), 0 ꢁC; (v) C6Cl5OH, DMAP, DCC, THF, 0 ꢁC;
(vi) H2NCHCONCH3OH,11 Et3N, THF, 25 ꢁC; (vii) H2/Pd, ethanol.

H. Ouchetto et al. / Bioorg. Med. Chem. 13 (2005) 1799–1803
1801
Figure 1. Fluorescence iron (III) titration curves with labelled ferri-
chrome analogue 1. Aliquots of stock solutions of 1 in MeOH were
treated with aliquots of methanolic solutions of FeCl3 (0, 0.2, 0.4, 0.6,
0.8 and 1 equiv) and diluted with MeOH (80%)–0.1 N aqueous NaOAc
(20%) to a final concentration of 5 lM.
Figure 2. Iron uptake by S. cerevisiae from either 55Fe-ferrichrome or
55Fe bound to ligand 1 (fluorescent analogue of ferrichrome). Cells
were grown overnight in YPD medium. Cells were then diluted 10-fold
in the same medium and cultured for 6 h before iron uptake was
measured from 1 lM ferrichrome (squares) or from 1 lM fluorescent
analogue of ferrichrome (1) (circles). Means SE from three
experiments.
scence of the NBD derivative 10 was not affected by
addition of similar concentrations of iron salts.
The selectivity of our probe has been tested in the pres-
ence of other biologically relevant metal ions such as
Ca(II), Mg(II) and Cu(II) metal ions. Ca2+ and Mg2+
had no effect on fluorescence signal of 1 and Cu2+ pro-
duced significant quenching but clearly less important
than with Fe3+
.
2.3. Biological activity
We first tested that the iron bound to the fluorescent
analogue of ferrichrome 1 was taken up by Saccharomy-
ces cerevisiae cells as efficiently as the iron bound to
commercially-available ferrichrome. Figure 2 shows that
the kinetics of iron uptake from both ligands are similar.
Then we checked that the fluorescent derivative of ferri-
chrome itself actually entered cells that are known to
have specific ferrichrome transport mechanisms: S. cere-
visiae12 and C. albicans13 Figure 3A and B shows that it
was indeed the case. In both S. cerevisiae (Fig. 3A) and
in C. albicans (Fig. 3B) cells, fluorescence accumulated
preferentially in peri-vacuolar vesicles, which is consis-
tent with the endocytosis-mediated mechanism of sidero-
phore uptake proposed by Kim et al.14 Cells that
appeared full of fluorescence (Fig. 3A) were probably
dead cells, freely permeable to the siderophore. Tritricho-
monas foetus is a pathogenic flagellate of the uro-geni-
tal tract of cattle that has an unusually high nutritional
requirement for iron.15 Siderophore used by this organ-
ism has never been shown. Figure 3C shows that T.
foetus accumulated 1 into small vesicles that could be
pinocytosis vesicles. An extensive study of siderophore
iron uptake/used by T. foetus will be published else-
where. Figure 3 shows that ligand 1 is a useful tool in
order to study ferrichrome uptake and used by various
organisms.
Figure 3. Confocal micrographs of S. cerevisiae (A), C. albicans (B)
and T. foetus (C) cells grown overnight in the presence of 10 lM of the
fluorescent analogue of ferrichrome (1).

1802
H. Ouchetto et al. / Bioorg. Med. Chem. 13 (2005) 1799–1803
3. Conclusion and prospects
The precipitate of dicyclohexylurea was eliminated by
filtration and the filtrate was concentrated to dryness.
The resulting residue was purified, first by filtration on
column (neutral alumina gel, AcOEt) then by column
chromatography (silica gel, AcOEt/petroleum ether
35:65), yielding 6 (2.20 g, 72%), a colourless visc-
ous oil, 1H NMR (500 MHz, CDCl3) d 2.91 (t, 6H,
J = 5.9 Hz), 3.77 (s, 6H), 3.84 (t, 6H, J = 5.9 Hz), 5.03
(s, 2H), 5.22 (s, 1H), 7.27 (m, 5H). MS (ESI+): m/z
1209 and good correlation with isotopic cluster.
A simple method to produce a ferrichrome-type sidero-
phore bearing a free amino group is reported. A fluores-
cent labelled artificial siderophore (1) was prepared by
linking a fluorescent nucleus to the amino group. This
compound was actually uptaken by the cell and was
used as a source of iron, like the ferrichrome itself, in
C. albicans and in S. cerevisiae but also in T. foetus
for which such a result has never been shown. This syn-
thetic fluorescent probe 1 is a promising tool for study-
ing siderophore uptake mechanisms as the intracellular
localization of the ligand but its behaviour in the cell
could also be monitored.
4.3. Benzyl N-Tris{[2-[((N-hydroxy-N-methylcarbamo-
yl)methyl)aminocarbonyl]ethoxy]methyl}methylcarba-
mate (7)
Compound 2, as easily derivatized as desferrioxamine B,
will be very useful in order to have access to new
biological tools. It could also be investigated as the
siderophore component of conjugates, particularly
siderophore-antifongic agent conjugates.
To a solution of 6 (4.95 g, 4.07 mmol) in THF (60 mL),
placed in a three necked round bottomed flask under N2,
was added a mixture of Et3N (2.47 g, 24.42 mmol) and
N-hydroxy-N-methylcarbamoylmethyl
ammonium
2,2,2-trifluoroacetate11 (5.33 g, 24.42 mmol) in suspen-
sion in THF (60 mL). The mixture was stirred at room
temperature for 4 days and the solvent was then evapo-
rated under reduced pressure. After addition of water
(100 mL), the aqueous layer was extracted with CH2Cl2
(100 mL) and the organic phase was washed with water
(3 · 50 mL). The aqueous phases were collected and
concentrated to dryness. The resulting residue was puri-
fied by column chromatography (silica) by using AcOEt/
MeOH (85:15) as eluent, giving 7 (1.45 g, 49%), an or-
4. Experimental
4.1. Chemistry
All reagents were analytical grade, dried and purified
when necessary.
1
TLC analyses were carried out on pre-coated plates of
silica or alumina gel 60 F254 (Merck). The visualization
was realized by using a two-wavelength (365 and
254 nm) detection system or iodine and, in the case of
the hydroxamate ligands, a FeCl3 solution. Silica gel
used for column chromatography was 60A granulome-
try 6–35 or 40–63 lm (SDS) or Florisilꢂ 100–200 mesh
(ACROS).
ange viscous oil, H NMR (270 MHz, CD3OD) d 2.48
(t, 6H, J = 5.9 Hz), 3.18 (s, 9H), 3.7 (s and t, 12H,
J = 5.9 Hz), 4.13 (s, 6H), 5.00 (s, 2H), 7.27–7.33 (m,
5H). 1H NMR (500 MHz, DMSO-d6) d 2.35 (t, 6H,
J = 6.4 Hz), 3.07 (s, 9H), 3.50 (s, 6H), 3.54 (t, 6H,
J = 6.4 Hz), 3.95 (d, 6H, J = 5.3 Hz), 4.96 (s, 2H), 6.56
(br s, 1H), 7.26–7.36 (m, 5H), 7.89 (t, 3H, J = 5 Hz),
9.91 (br s, 3H). MS (ESIꢀ): m/z 728 (MꢀH)ꢀ, 842
(MꢀH+CF3CO2H)ꢀ.
Spectra were obtained as follows: 1H NMR spectra were
recorded at 270 MHz on a Jeol GSX 270 WB spectro-
meter or at 500 MHz on a Brucker AVANCE DRX
500 and 13C NMR spectra were recorded at
125.75 MHz on the Brucker AVANCE DRX; FTIR
spectra were recorded on a BIO-RAD spectrometer;
ESI-MS spectra were recorded on a JMS-700 (JEOL
LTD, Akishima, Tokyo, Japan) double focusing mass
spectrometer with reversed geometry, equipped with a
pneumatically assisted electrospray ionization (ESI)
source and MALDI-TOF-MS spectra on a Brucker
BIFLEX III.
4.4. Tris{[2-[((N-hydroxy-N-methylcarbamoyl)methyl)-
aminocarbonyl]ethoxy]methyl} methylamine (2)
Compound 7 (1.02g, 1.40 mmol) was stirred with of
10% Pd/C (122 mg) in ethanol (100 mL) under H2 at
room temperature for 4 h 30min. The palladium was fil-
tered off and the solvent removed under reduced pres-
sure, giving amine 2 (0.83 g, 100%), as a hygroscopic
1
light yellow solid, H NMR (500 MHz, DMSO-d6) d
2.36 (t, 6H, J = 6.3 Hz), 3.07 (s, 9H), 3.17 (s, 6H), 3.55
(t, 6H, J = 6.3 Hz), 3.95 (d, 6H, J = 5.2Hz), 7.91 (br s,
3H), 10.05 (br s, 3H). H NMR (500 MHz, CD3 OD)
1
Fluorescence experiments were performed by using a
QM-4/QuantaMasterTM fluorometer from PTIꢂ.
d 2.37 (t, 6H, J = 5.8 Hz), 3.19 (s, 9H), 3.42(s, 6H),
3.70 (t, 6H, J = 5.8 Hz), 4.14 (s, 6H). 13C NMR
(125.75 MHz, DMSO-d6) d 35.7, 39.7, 56.6, 67.2, 71.2,
168.9, 170.3. MS (MALDI TOF): m/z 596 (M + H)+,
618 (M+Na)+.
4.2. Benzyl N-Tris{[2-(2,3,4,5,6-pentachlorophenoxycar-
bonyl)ethoxy]methyl}methyl carbamate (6)
Triacid
5
(1.19 g, 2.52 mmol), pentachlorophenol
4.5. 4-[4-(7-Nitro-2,1,3-benzoxadiazol-4-yl)piperazino]-4-
oxobutanoic acid (10)
(2.68 g, 10.09 mmol) and DMAP (24.6 mg, 0.20 mmol)
were dissolved in THF (20 mL). The solution was cooled
to 0 ꢁC and a solution of DCC (2.08 g, 10.09 mmol) in
THF (20 mL) was then added. The reaction mixture
was stirred under N2 at room temperature for 48 h.
4-Nitro-7-piperazino-2,1,3-benzoxadiazole5 (0.50 g, 2.0
mmol) and succinic anhydride (0.22 g, 2.2 mmol) were
dissolved in toluene (30 mL) and DMF (10 mL). The

H. Ouchetto et al. / Bioorg. Med. Chem. 13 (2005) 1799–1803
1803
solution was refluxed overnight. After removal of the
solvents under reduced pressure, the residue was purified
by column chromatography (silica gel, 97:3 CH2Cl2/
MeOH) yielded a red solid (0.65 g, 92%) identified to
cose). T. foetus was grown on Trypticase, yeast extract,
maltose medium (TYM).17 For microscopy experiments,
cells were grown overnight in the presence of 10 lM of
the ferri-siderophore and then washed with water (S.
cerevisiae, C. albicans) or with isotonic buffer (T. foetus).
1
10, H NMR (270 MHz, DMSO-d6) d 2.44 (t, 2H, J =
6.4 Hz), 2.59 (t, 2H, J = 6.4 Hz), 3.73–4.24 (4t, 8H,
J = 5.1 Hz), 6.59 (d, 1H, J = 9 Hz), 8.50 (d, 1H,
J = 9 Hz). MS (MALDI TOF): m/z 372(M+Na) +, 394
(M+2NaꢀH)+.
Iron uptake assays were performed as previously de-
scribed12 by using commercial ferrichrome (Sigma) or
the newly synthesized fluorescent derivative of ferri-
chrome labelled with 55Fe.
4.6. N-Succinimidyl-4-[4-(7-nitro-2,1,3-benzoxadiazol-4-
yl)piperazino]-4-oxobutanoate (8)
Acknowledgements
Fluorescent acid 10 (200 mg, 0.572 mmol), NHS
(65.8 mg, 0.572mmol) and DCC (118 mg, 0.573 mmol)
were dissolved in THF (8 mL) and DMF (6 mL). The
reaction mixture was stirred under N2 at room temper-
ature for 2days. After removal of the precipitate by fil-
tration, the filtrate was concentrated to dryness under
reduced pressure. The resulting residue was purified by
column chromatography (silica gel, AcOEt) giving ester
8 (0.17 g, 67%), a red solid, 1H NMR (270 MHz,
The authors thank Dr. F. X. Sauvage for a critical read-
ing of the manuscript. This work was supported by the
`
´
Ministere de la Recherche et de lÕEnseignement Superi-
eur (Programme de Recherches Fondamentales en
Microbiologie et Maladies Infectieuses).
References and notes
DMSO-d6)
d
2.74–2.79 (m, 6H), 2.90 (t, 2H,
J = 6.6 Hz), 3.73–4.24 (4 m, 8H, J = 5.1 Hz), 6.60 (d,
1H, J = 9.1 Hz), 8.51 (d, 1H, J = 9.1 Hz). MS (MALDI
TOF): m/z 469 (M+Na)+.
1. Neilands, J. B. Ann. Rev. Biochem. 1981, 50, 715.
2. Neilands, J. B.; Konopka, K.; Schwyn, B.; Coy, M.;
Francis, R. T.; Paw, B. H.; Bagg, A. In Iron Transport in
Microbes, Plants, and Animals; Winkelmann, G., van der
Helm, D., Neilands, J. B., Eds. Weinheim, 1987; pp 3–33.
3. Lesuisse, E.; Knight, S. A. B.; Camadro, J.-M.; Dancis, A.
Yeast 2002, 19, 329.
4. Weizman, H.; Ardon, O.; Mester, B.; Libman, J.; Dwir,
O.; Hadar, Y.; Chen, Y.; Shanzer, A. J. Am. Chem. Soc.
1996, 118, 12368.
4.7. 4-[4-(7-Nitro-2,1,3-benzoxadiazol-4-yl)piperazino]-
oxopropylN-tris{[2-[((N-hydroxy-N-methylcarba-
moyl)methyl)aminocarbonyl]ethoxy]methyl}methylamide
(1)
Amine 2 (120 mg, 0.20 mmol) and ester 8 (108 mg,
0.24 mmol) were dissolved in THF (6 mL) and DMF
(2.5 mL). The solution was stirred under N2 at room
temperature for 4 days then the solvents were evapo-
rated under reduced pressure. The resulting residue
was purified by column chromatography (silica gel,
85:15 CH2Cl2/MeOH), yielding 1, a red viscous oil
5. Nudelman, R.; Ardon, O.; Hadar, Y.; Chen, Y.; Libman,
J.; Shanzer, A. J. Med. Chem. 1998, 41, 1671.
6. Lytton, S. D.; Cabantchik, Z. I.; Libman, J.; Shanzer, A.
Mol. Pharmacol. 1991, 40, 584.
7. Nelson, M.; Carrano, C. J.; Szaniszlo, P. J. BioMetals
1992, 5, 37.
8. Ghosh, M.; Lambert, L. J.; Huber, P. W.; Miller, M. J.
Bioorg. Med. Chem. Lett. 1995, 5, 2337.
9. Newkome, G. R.; Lin, X.; Young, J. K. Synlett 1992,
53.
10. Cardona, C. M.; Gawley, R. E. J. Org. Chem. 2002, 67,
1411.
11. Dayan, I.; Libman, J.; Agi, Y.; Shanzer, A. Inorg. Chem.
1993, 32, 1467.
12. Lesuisse, E.; Blaiseau, P. L.; Dancis, A.; Camadro, J.-M.
Microbiology 2001, 147, 289.
13. Ardon, O.; Bussy, H.; Philpott, C.; Ward, D. M.; Davis-
Kaplan, S.; Verroneau, S.; Bojiang; Kaplan, J. J. Biol.
Chem. 2001, 276, 43049.
1
(38 mg, 20%), H NMR (500 MHz, CD3 OD) d 2.50
(t, 6H, J = 5.8 Hz), 2.53 (t, 2H, J = 6.7 Hz), 2.68 (t,
2H, J = 6.7 Hz), 3.19 (s, 9H), 3.68 (t, 6H, J = 5.9 Hz),
3.72(s, 6H), 4.15 (s, 6H), 4.28–3.88 (m, 8H, J = 5 Hz),
6.48 (d, 1H, J = 8,9 Hz), 8.42(d, 1H, J = 8.9 Hz). 13C
NMR (125.75 MHz, CD3OD) d 29.3, 32.4, 36.4, 37.4,
41.4, 42.0, 45.3, 61.5, 68.5, 70.2, 104.0, 124.0, 136.7,
146.0, 146.3, 146.5, 170.9, 173.3, 174.4, 174.9.IR
(KBr): m 3421, 1648, 1553 cmꢀ1. MS (MALDI-TOF):
m/z 949 (MꢀH+Na)+, 950 (M+Na)+.
14. Kim, Y.; Yun, C. W.; Philpott, C. C. EMBO J. 2002, 21,
3632.
4.8. Biological studies
15. Tachezy, J.; Suchan, P.; Schrevel, J.; Kulda, J. Exp.
Parasitol. 1998, 90, 155.
16. Ardon, O.; Nudelman, R.; Caris, C.; Libman, J.; Shanzer,
A.; Chen, Y.; Hadar, Y. J. Bacteriol. 1998, 180, 2021.
17. Diamond, L. S. J. Parasitol. 1957, 43, 488.
Fluorescence confocal microscopy was performed as de-
scribed by Ardon et al.16 C. albicans (strain BWP17) and
S. cerevisiae (strain BY4741) were grown on complete
YPD medium (1% yeast extract, 1% peptone, 2% glu-